Micro-costing of genetic diagnostics in acute leukemia in Sweden: from standard-of-care to whole-genome sequencing.

AIMS AND BACKGROUND: Whole-genome sequencing (WGS) is increasingly applied in clinical practice and expected to replace standard-of-care (SoC) genetic diagnostics in hematological malignancies. This study aims to assess and compare the fully burdened cost ('micro-costing') per patient for Swedish laboratories using WGS and SoC, respectively, in pediatric and adult patients with acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). METHODS: The resource use and cost details associated with SoC, e.g. chromosome banding analysis, fluorescent in situ hybridization, and targeted sequencing analysis, were collected via activity-based costing methods from four diagnostic laboratories. For WGS, corresponding data was collected from two of the centers. A simulation-based scenario model was developed for analyzing the WGS cost based on different annual sample throughput to evaluate economy of scale. RESULTS: The average SoC total cost per patient was €2,465 for pediatric AML and €2,201 for pediatric ALL, while in adults, the corresponding cost was €2,458 for AML and €1,207 for ALL. The average WGS cost (90x tumor/30x normal; sequenced on the Illumina NovaSeq 6000 platform) was estimated to €3,472 based on an annual throughput of 2,500 analyses, however, with an annual volume of 7,500 analyses the average cost would decrease by 23% to €2,671. CONCLUSION: In summary, WGS is currently more costly than SoC, however the cost can be reduced by utilizing laboratories with higher throughput and by the expected decline in cost of reagents. Our data provides guidance to decision-makers for the resource allocation needed when implementing WGS in diagnostics of hematological malignancies.

Precision Diagnostics in Myeloid Malignancies: Development and Validation of a National Capture-Based Gene Panel.

Gene panel sequencing has become a common diagnostic tool for detecting somatically acquired mutations in myeloid neoplasms. However, many panels have restricted content, provide insufficient sensitivity levels, or lack clinically validated workflows. We here describe the development and validation of the Genomic Medicine Sweden myeloid gene panel (GMS-MGP), a capture-based 191 gene panel including mandatory genes in contemporary guidelines as well as emerging candidates. The GMS-MGP displayed uniform coverage across all targets, including recognized difficult GC-rich areas. The validation of 117 previously described somatic variants showed a 100% concordance with a limit-of-detection of a 0.5% variant allele frequency (VAF), achieved by utilizing error correction and filtering against a panel-of-normals. A national interlaboratory comparison investigating 56 somatic variants demonstrated highly concordant results in both detection rate and reported VAFs. In addition, prospective analysis of 323 patients analyzed with the GMS-MGP as part of standard-of-care identified clinically significant genes as well as recurrent mutations in less well-studied genes. In conclusion, the GMS-MGP workflow supports sensitive detection of all clinically relevant genes, facilitates novel findings, and is, based on the capture-based design, easy to update once new guidelines become available. The GMS-MGP provides an important step toward nationally harmonized precision diagnostics of myeloid malignancies.

Multimodal classification of molecular subtypes in pediatric acute lymphoblastic leukemia.

Genomic analyses have redefined the molecular subgrouping of pediatric acute lymphoblastic leukemia (ALL). Molecular subgroups guide risk-stratification and targeted therapies, but outcomes of recently identified subtypes are often unclear, owing to limited cases with comprehensive profiling and cross-protocol studies. We developed a machine learning tool (ALLIUM) for the molecular subclassification of ALL in retrospective cohorts as well as for up-front diagnostics. ALLIUM uses DNA methylation and gene expression data from 1131 Nordic ALL patients to predict 17 ALL subtypes with high accuracy. ALLIUM was used to revise and verify the molecular subtype of 281 B-cell precursor ALL (BCP-ALL) cases with previously undefined molecular phenotype, resulting in a single revised subtype for 81.5% of these cases. Our study shows the power of combining DNA methylation and gene expression data for resolving ALL subtypes and provides a comprehensive population-based retrospective cohort study of molecular subtype frequencies in the Nordic countries.

Feasibility to use whole-genome sequencing as a sole diagnostic method to detect genomic aberrations in pediatric B-cell acute lymphoblastic leukemia.

Introduction: The suitability of whole-genome sequencing (WGS) as the sole method to detect clinically relevant genomic aberrations in B-cell acute lymphoblastic leukemia (ALL) was investigated with the aim of replacing current diagnostic methods. Methods: For this purpose, we assessed the analytical performance of 150 bp paired-end WGS (90x leukemia/30x germline). A set of 88 retrospective B-cell ALL samples were selected to represent established ALL subgroups as well as ALL lacking stratifying markers by standard-of-care (SoC), so-called B-other ALL. Results: Both the analysis of paired leukemia/germline (L/N)(n=64) as well as leukemia-only (L-only)(n=88) detected all types of aberrations mandatory in the current ALLTogether trial protocol, i.e., aneuploidies, structural variants, and focal copy-number aberrations. Moreover, comparison to SoC revealed 100% concordance and that all patients had been assigned to the correct genetic subgroup using both approaches. Notably, WGS could allocate 35 out of 39 B-other ALL samples to one of the emerging genetic subgroups considered in the most recent classifications of ALL. We further investigated the impact of high (90x; n=58) vs low (30x; n=30) coverage on the diagnostic yield and observed an equally perfect concordance with SoC; low coverage detected all relevant lesions. Discussion: The filtration of the WGS findings with a short list of genes recurrently rearranged in ALL was instrumental to extract the clinically relevant information efficiently. Nonetheless, the detection of DUX4 rearrangements required an additional customized analysis, due to multiple copies of this gene embedded in the highly repetitive D4Z4 region. We conclude that the diagnostic performance of WGS as the standalone method was remarkable and allowed detection of all clinically relevant genomic events in the diagnostic setting of B-cell ALL.

Establishment and characterization of a novel breast implant-associated anaplastic large cell lymphoma cell line and PDX model (BIA-XR1) with a unique KRAS mutation.

Breast implant-associated anaplastic large cell lymphoma (BIA-ALCL) is an uncommon T-cell lymphoma type with distinct clinical, molecular and genetic features. Establishment of BIA-ALCL cell lines and patient-derived xenograft (PDX) models are essential experimental tools to investigate the molecular pathogenesis of the disease. We characterized a novel BIA-ALCL cell line and PDX model, named BIA-XR1, derived from a patient with textured breast implant who developed lymphoma. Next-generation sequencing revealed a STAT3 mutation, commonly detected in BIA-ALCL, and a unique KRAS mutation reported for the first time in this lymphoma type. Both JAK/STAT3 and RAS/MEK/ERK oncogenic pathways were activated in BIA-XR1, which are targetable with clinically available agents.

The prognostic impact of IKZF1 deletions and UKALL genetic classifiers in paediatric B-cell precursor acute lymphoblastic leukaemia treated according to NOPHO 2008 protocols.

We investigated 390 paediatric B-cell precursor acute lymphoblastic leukaemia (BCP-ALL) patients treated according to NOPHO ALL 2008, regarding copy number alterations (CNA) of eight loci associated with adverse prognosis, including IKZF1. The impact on outcome was investigated for each locus individually, combined as CNA profiles and together with cytogenetic information. The presence of IKZF1 deletion or a poor-risk CNA profile was associated with poor outcome in the whole cohort. In the standard-risk group, IKZF1-deleted cases had an inferior probability of relapse-free survival (pRFS) (p ≤ 0.001) and overall survival (pOS) (p ≤ 0.001). Additionally, among B-other patients, IKZF1 deletion correlated with poor pRFS (60% vs. 90%) and pOS (65% vs. 89%). Both IKZF1 deletion and a poor-risk CNA profile were independent factors for relapse and death in multivariable analyses adjusting for known risk factors including measurable residual disease. Our data indicate that BCP-ALL patients with high-risk CNA or IKZF1 deletion have worse prognosis despite otherwise low-risk features. Conversely, patients with both a good CNA and cytogenetic profile had a superior relapse-free (p ≤ 0.001) and overall survival (p ≤ 0.001) in the cohort, across all risk groups. Taken together, our findings highlight the potential of CNA assessment to refine stratification in ALL.

Simultaneous Ultra-Sensitive Detection of Structural and Single Nucleotide Variants Using Multiplex Droplet Digital PCR in Liquid Biopsies from Children with Medulloblastoma.

Medulloblastoma is a malignant embryonal tumor of the central nervous system (CNS) that mainly affects infants and children. Prognosis is highly variable, and molecular biomarkers for measurable residual disease (MRD) detection are lacking. Analysis of cell-free DNA (cfDNA) in cerebrospinal fluid (CSF) using broad genomic approaches, such as low-coverage whole-genome sequencing, has shown promising prognostic value. However, more sensitive methods are needed for MRD analysis. Here, we show the technical feasibility of capturing medulloblastoma-associated structural variants and point mutations simultaneously in cfDNA using multiplexed droplet digital PCR (ddPCR). Assay sensitivity was assessed with a dilution series of tumor in normal genomic DNA, and the limit of detection was below 100 pg of input DNA for all assays. False positive rates were zero for structural variant assays. Liquid biopsies (CSF and plasma, n = 47) were analyzed from 12 children with medulloblastoma, all with negative CSF cytology. MRD was detected in 75% (9/12) of patients overall. In CSF samples taken before or within 21 days of surgery, MRD was detected in 88% (7/8) of patients with localized disease and in one patient with the metastasized disease. Our results suggest that this approach could expand the utility of ddPCR and complement broader analyses of cfDNA for MRD detection.

The clinical picture of ERCC6L2 disease: from bone marrow failure to acute leukemia.

Biallelic germ line excision repair cross-complementing 6 like 2 (ERCC6L2) variants strongly predispose to bone marrow failure (BMF) and myeloid malignancies, characterized by somatic TP53-mutated clones and erythroid predominance. We present a series of 52 subjects (35 families) with ERCC6L2 biallelic germ line variants collected retrospectively from 11 centers globally, with a follow-up of 1165 person-years. At initial investigations, 32 individuals were diagnosed with BMF and 15 with a hematological malignancy (HM). The subjects presented with 19 different variants of ERCC6L2, and we identified a founder mutation, c.1424delT, in Finnish patients. The median age of the subjects at baseline was 18 years (range, 2-65 years). Changes in the complete blood count were mild despite severe bone marrow (BM) hypoplasia and somatic TP53 mutations, with no significant difference between subjects with or without HMs. Signs of progressive disease included increasing TP53 variant allele frequency, dysplasia in megakaryocytes and/or erythroid lineage, and erythroid predominance in the BM morphology. The median age at the onset of HM was 37.0 years (95% CI, 31.5-42.5; range, 12-65 years). The overall survival (OS) at 3 years was 95% (95% CI, 85-100) and 19% (95% CI, 0-39) for patients with BMF and HM, respectively. Patients with myelodysplastic syndrome or acute myeloid leukemia with mutated TP53 undergoing hematopoietic stem cell transplantation had a poor outcome with a 3-year OS of 28% (95% CI, 0-61). Our results demonstrated the importance of early recognition and active surveillance in patients with biallelic germ line ERCC6L2 variants.

Duplex Sequencing Uncovers Recurrent Low-frequency Cancer-associated Mutations in Infant and Childhood KMT2A-rearranged Acute Leukemia.

Infant acute lymphoblastic leukemia (ALL) with KMT2A-gene rearrangements (KMT2A-r) have few mutations and a poor prognosis. To uncover mutations that are below the detection of standard next-generation sequencing (NGS), a combination of targeted duplex sequencing and NGS was applied on 20 infants and 7 children with KMT2A-r ALL, 5 longitudinal and 6 paired relapse samples. Of identified nonsynonymous mutations, 87 had been previously implicated in cancer and targeted genes recurrently altered in KMT2A-r leukemia and included mutations in KRAS, NRAS, FLT3, TP53, PIK3CA, PAX5, PIK3R1, and PTPN11, with infants having fewer such mutations. Of identified cancer-associated mutations, 62% were below the resolution of standard NGS. Only 33 of 87 mutations exceeded 2% of cellular prevalence and most-targeted PI3K/RAS genes (31/33) and typically KRAS/NRAS. Five patients only had low-frequency PI3K/RAS mutations without a higher-frequency signaling mutation. Further, drug-resistant clones with FLT3 D835H or NRAS G13D/G12S mutations that comprised only 0.06% to 0.34% of diagnostic cells, expanded at relapse. Finally, in longitudinal samples, the relapse clone persisted as a minor subclone from diagnosis and through treatment before expanding during the last month of disease. Together, we demonstrate that infant and childhood KMT2A-r ALL harbor low-frequency cancer-associated mutations, implying a vast subclonal genetic landscape.

Patient-Specific Assays Based on Whole-Genome Sequencing Data to Measure Residual Disease in Children With Acute Lymphoblastic Leukemia: A Proof of Concept Study.

Risk-adapted treatment in acute lymphoblastic leukemia (ALL) relies on genetic information and measurable residual disease (MRD) monitoring. In this proof of concept study, DNA from diagnostic bone marrow (BM) of six children with ALL, without stratifying genetics or central nervous system (CNS) involvement, underwent whole-genome sequencing (WGS) to identify structural variants (SVs) in the leukemic blasts. Unique sequences generated by SVs were targeted with patient-specific droplet digital PCR (ddPCR) assays. Genomic DNA (gDNA) from BM and cell-free DNA (cfDNA) from plasma and cerebrospinal fluid (CSF) were analyzed longitudinally. WGS with 30× coverage enabled target identification in all cases. Limit of quantifiability (LoQ) and limit of detection (LoD) for the ddPCR assays (n = 15) were up to 10-5 and 10-6, respectively. All targets were readily detectable in a multiplexed ddPCR with minimal DNA input (1 ng of gDNA) at a 10-1 dilution, and targets for half of the patients were also detectable at a 10-2 dilution. The level of MRD in BM at end of induction and end of consolidation block 1 was in a comparable range between ddPCR and clinical routine methods for samples with detectable residual disease, although our approach consistently detected higher MRD values for patients with B-cell precursor ALL. Additionally, several samples with undetectable MRD by flow cytometry were MRD-positive by ddPCR. In plasma, the level of leukemic targets decreased in cfDNA over time following the MRD level detected in BM. cfDNA was successfully extracted from all diagnostic CSF samples (n = 6), and leukemic targets were detected in half of these. The results suggest that our approach to design molecular assays, together with ddPCR quantification, is a technically feasible option for accurate MRD quantification and that cfDNA may contribute valuable information regarding MRD and low-grade CNS involvement.

Standardization of molecular monitoring of CML: results and recommendations from the European treatment and outcome study.

Standardized monitoring of BCR::ABL1 mRNA levels is essential for the management of chronic myeloid leukemia (CML) patients. From 2016 to 2021 the European Treatment and Outcome Study for CML (EUTOS) explored the use of secondary, lyophilized cell-based BCR::ABL1 reference panels traceable to the World Health Organization primary reference material to standardize and validate local laboratory tests. Panels were used to assign and validate conversion factors (CFs) to the International Scale and assess the ability of laboratories to assess deep molecular response (DMR). The study also explored aspects of internal quality control. The percentage of EUTOS reference laboratories (n = 50) with CFs validated as optimal or satisfactory increased from 67.5% to 97.6% and 36.4% to 91.7% for ABL1 and GUSB, respectively, during the study period and 98% of laboratories were able to detect MR4.5 in most samples. Laboratories with unvalidated CFs had a higher coefficient of variation for BCR::ABL1IS and some laboratories had a limit of blank greater than zero which could affect the accurate reporting of DMR. Our study indicates that secondary reference panels can be used effectively to obtain and validate CFs in a manner equivalent to sample exchange and can also be used to monitor additional aspects of quality assurance.

A Study Protocol for Validation and Implementation of Whole-Genome and -Transcriptome Sequencing as a Comprehensive Precision Diagnostic Test in Acute Leukemias.

Background: Whole-genome sequencing (WGS) and whole-transcriptome sequencing (WTS), with the ability to provide comprehensive genomic information, have become the focal point of research interest as novel techniques that can support precision diagnostics in routine clinical care of patients with various cancer types, including hematological malignancies. This national multi-center study, led by Genomic Medicine Sweden, aims to evaluate whether combined application of WGS and WTS (WGTS) is technically feasible and can be implemented as an efficient diagnostic tool in patients with acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). In addition to clinical impact assessment, a health-economic evaluation of such strategy will be performed. Methods and Analysis: The study comprises four phases (i.e., retrospective, prospective, real-time validation, and follow-up) including approximately 700 adult and pediatric Swedish AML and ALL patients. Results of WGS for tumor (90×) and normal/germline (30×) samples as well as WTS for tumors only will be compared to current standard of care diagnostics. Primary study endpoints are diagnostic efficiency and improved diagnostic yield. Secondary endpoints are technical and clinical feasibility for routine implementation, clinical utility, and health-economic impact. Discussion: Data from this national multi-center study will be used to evaluate clinical performance of the integrated WGTS diagnostic workflow compared with standard of care. The study will also elucidate clinical and health-economic impacts of a combined WGTS strategy when implemented in routine clinical care. Clinical Trial Registration: [https://doi.org/10.1186/ISRCTN66987142], identifier [ISRCTN66987142].

A somatic UBA2 variant preceded ETV6-RUNX1 in the concordant BCP-ALL of monozygotic twins.

Genetic analysis of leukemic clones in monozygotic twins with concordant acute lymphoblastic leukemia (ALL) has proved a unique opportunity to gain insight into the molecular phylogenetics of leukemogenesis. Using whole-genome sequencing, we characterized constitutional and somatic single nucleotide variants/insertion-deletions (indels) and structural variants in a monozygotic twin pair with concordant ETV6-RUNX1+ B-cell precursor ALL (BCP-ALL). In addition, digital PCR (dPCR) was applied to evaluate the presence of and quantify selected somatic variants at birth, diagnosis, and remission. A shared somatic complex rearrangement involving chromosomes 11, 12, and 21 with identical fusion sequences in leukemias of both twins offered direct proof of a common clonal origin. The ETV6-RUNX1 fusion detected at diagnosis was found to originate from this complex rearrangement. A shared somatic frameshift deletion in UBA2 was also identified in diagnostic samples. In addition, each leukemia independently acquired analogous deletions of 3 genes recurrently targeted in BCP-ALLs (ETV6, ATF7IP, and RAG1/RAG2), providing evidence of a convergent clonal evolution only explained by a strong concurrent selective pressure. Quantification of the UBA2 deletion by dPCR surprisingly indicated it persisted in remission. This, for the first time to our knowledge, provided evidence of a UBA2 variant preceding the well-established initiating event ETV6-RUNX1. Further, we suggest the UBA2 deletion exerted a leukemia predisposing effect and that its essential role in Small Ubiquitin-like Modifier (SUMO) attachment (SUMOylation), regulating nearly all physiological and pathological cellular processes such as DNA-repair by nonhomologous end joining, may hold a mechanistic explanation for the predisposition.

Case Report: Whole genome sequencing identifies CCDC88C as a novel JAK2 fusion partner in pediatric T-cell acute lymphoblastic leukemia.

In the present report, we applied whole genome sequencing (WGS) to genetically characterize a case of pediatric T-cell acute lymphoblastic leukemia (ALL) refractory to standard therapy. WGS identified a novel JAK2 fusion, with CCDC88C as a partner. CCDC88C encodes a protein part of the Wnt signaling pathway and has previously been described in hematological malignancies as fusion partner to FLT3 and PDGFRB. The novel CCDC88C::JAK2 fusion gene results in a fusion transcript, predicted to produce a hybrid protein, which retains the kinase domain of JAK2 and is expected to respond to JAK2 inhibitors. This report illustrates the potential of WGS in the diagnostic setting of ALL.

Minimal residual disease, long-term outcome, and IKZF1 deletions in children and adolescents with Down syndrome and acute lymphocytic leukaemia: a matched cohort study.

BACKGROUND: Patients with Down syndrome and acute lymphocytic leukaemia are at an increased risk of treatment-related mortality and relapse, which is influenced by unfavourable genetic aberrations (eg, IKZF1 deletion). We aimed to investigate the potential underlying effect of Down syndrome versus the effects of adverse cancer genetics on clinical outcome. METHOD: Patients (aged 1-23 years) with Down syndrome and acute lymphocytic leukaemia and matched non-Down syndrome patients with acute lymphocytic leukaemia (matched controls) from eight trials (DCOG ALL10 and ALL11, ANZCHOG ALL8, AIEOP-BFM ALL2009, UKALL2003, NOPHO ALL2008, CoALL 07-03, and CoALL 08-09) done between 2002 and 2018 across various countries (the Netherlands, the UK, Australia, Denmark, Finland, Iceland, Norway, Sweden, and Germany) were included. Participants were matched (1:3) for clinical risk factors and genetics, including IKZF1 deletion. The primary endpoint was the comparison of MRD levels (absolute MRD levels were categorised into two groups, low [<0·0001] and high [≥0·0001]) between patients with Down syndrome and acute lymphocytic leukaemia and matched controls, and the secondary outcomes were comparison of long-term outcomes (event-free survival, overall survival, relapse, and treatment-related mortality [TRM]) between patients with Down syndrome and acute lymphocytic leukaemia and matched controls. Two matched cohorts were formed: for MRD analyses and for long-term outcome analyses. For both cohorts, matching was based on induction regimen; for the long-term outcome cohort, matching also included MRD-guided treatment group. We used mixed-effect models, Cox models, and competing risk for statistical analyses. FINDINGS: Of 251 children and adolescents with Down syndrome and acute lymphocytic leukaemia, 136 were eligible for analyses and matched to 407 (of 8426) non-Down syndrome patients with acute lymphocytic leukaemia (matched controls). 113 patients with Down syndrome and acute lymphocytic leukaemia were excluded from matching in accordance with predefined rules, no match was available for two patients with Down syndrome and acute lymphocytic leukaemia. The proportion of patients with high MRD at the end of induction treatment was similar for patients with Down syndrome and acute lymphocytic leukaemia (52 [38%] of 136) and matched controls (157 [39%] of 403; OR 0·97 [95% CI 0·64-1·46]; p=0·88). Patients with Down syndrome and acute lymphocytic leukaemia had a higher relapse risk than did matched controls in the IKZF1 deleted group (relapse at 5 years 37·1% [17·1-57·2] vs 13·2% [6·1-23·1]; cause-specific hazard ratio [HRcs] 4·3 [1·6-11·0]; p=0·0028), but not in the IKZF1 wild-type group (relapse at 5 years 5·8% [2·1-12·2] vs 8·1% [5·1-12·0]; HRcs 1·0 [0·5-2·1]; p=0·99). In addition to increased induction deaths (15 [6%] of 251 vs 69 [0·8%] of 8426), Down syndrome and acute lymphocytic leukaemia was associated with a higher risk of post-induction TRM compared with matched controls (TRM at 5 years 12·2% [7·0-18·9] vs 2·7% [1·3-4·9]; HRcs 5·0 [2·3-10·8]; p<0·0001). INTERPRETATION: Induction treatment is equivalently effective for patients with Down syndrome and acute lymphocytic leukaemia and for matched patients without Down syndrome. Down syndrome itself provides an additional risk in individuals with IKZF1 deletions, suggesting an interplay between the germline environment and this poor risk somatic aberration. Different treatment strategies are warranted considering both inherent risk of relapse and high risk of TRM. FUNDING: Stichting Kinder Oncologisch Centrum Rotterdam and the Princess Máxima Center Foundation, NHMRC Australia, The Cancer Council NSW, Tour de Cure, Blood Cancer UK, UK Medical Research Council, Children with Cancer, Swedish Society for Pediatric Cancer, Swedish Childhood Cancer Fund, Danish Cancer Society and the Danish Childhood Cancer Foundation.

Cytogenetic aberrations in adult acute lymphoblastic leukemia-A population-based study.

Cytogenetic aberrations are recognized as important prognostic factors in adult acute lymphoblastic leukemia (ALL), but studies seldom include elderly patients. From the population-based Swedish ALL Registry, we identified 728 patients aged 18-95 years, who were diagnosed with ALL 1997-2015 and had cytogenetic information. Registry data were complemented with original cytogenetic reports. BCR-ABL1 was the most recurrent aberration, with a frequency of 26%, with additional cytogenetic alterations in 64%. KTM2A rearrangement was the second most frequent aberration found in 7%. Low hypodiploidy-near triploidy and complex karyotype had negative impact, while t(1;19);TCF3-PBX1 showed positive impact on overall survival. However, after correction for age only complex karyotype remained significant.

Genomic arrays identify high-risk chronic lymphocytic leukemia with genomic complexity: a multi-center study.

Complex karyotype (CK) identified by chromosome-banding analysis (CBA) has shown prognostic value in chronic lymphocytic leukemia (CLL). Genomic arrays offer high-resolution genome-wide detection of copy-number alterations (CNAs) and could therefore be well equipped to detect the presence of a CK. Current knowledge on genomic arrays in CLL is based on outcomes of single center studies, in which different cutoffs for CNA calling were used. To further determine the clinical utility of genomic arrays for CNA assessment in CLL diagnostics, we retrospectively analyzed 2293 arrays from 13 diagnostic laboratories according to established standards. CNAs were found outside regions captured by CLL FISH probes in 34% of patients, and several of them including gains of 8q, deletions of 9p and 18p (p<0.01) were linked to poor outcome after correction for multiple testing. Patients (n=972) could be divided in three distinct prognostic subgroups based on the number of CNAs. Only high genomic complexity (high-GC), defined as ≥5 CNAs emerged as an independent adverse prognosticator on multivariable analysis for time to first treatment (Hazard ratio: 2.15, 95% CI: 1.36-3.41; p=0.001) and overall survival (Hazard ratio: 2.54, 95% CI: 1.54-4.17; p<0.001; n=528). Lowering the size cutoff to 1 Mb in 647 patients did not significantly improve risk assessment. Genomic arrays detected more chromosomal abnormalities and performed at least as well in terms of risk stratification compared to simultaneous chromosome banding analysis as determined in 122 patients. Our findings highlight genomic array as an accurate tool for CLL risk stratification.